Multiple human mycoplasma species cause chronic and opportunistic infections in both healthy and immunocompromised hosts. Mycoplasma hominis, the first human-associated mycoplasma to be identified, is widely associated with urogenital tract colonization that can lead to bacterial vaginosis, pelvic inflammatory disease and fever (post-partum or post-abortion). Extragenital tract infections including chronic arthritis osteomyelitis, assorted complications in newborn infants and disseminated infections have also been reported, which in immunocompromised hosts are potentially fatal. Our understanding of pathogenicity factors and strategies for host adaptation employed by M. hominis during arthritogenesis are largely unexplored. Furthermore, in cases of chronic arthritis, little is known of the underlying genetic changes that permit long term colonization despite the challenges of repeated antimicrobial therapy and the host immune system. Our knowledge base is currently limited by the lack of a defined genetic system for this pathogen, the absence of genome sequence information for any arthritogenic isolate and only two genomic datasets available for any isolates of the species. Accordingly, comparative genome sequencing is proposed for (i) a longitudinal series of twelve M. hominis isolates that were recovered over a 5-year period from a single patient with septic arthritis and (ii) a panel of twelve clinical isolates including an additional six serotypes, two urethritis strains from different continents, two wound-associated isolates, a tetracycline resistant isolate and a clinical isolate for which gene transfer has been reported. Analysis of the expression of two surface lipoproteins in the longitudinal series has demonstrated high frequency antigenic variation by insertion/deletion (indel) mutation within a homopolymeric tract and a novel mechanism of gene expression whereby a large, 40-kb genome duplication results in juxtaposition of a surface antigen gene with a rRNA gene promoter. Although such bottom up approaches have provided significant insight into surface antigen variation, two of the Specific Aims of this proposal are designed to use a top down strategy employing next generation sequencing to comprehensively delineate mutations and genomic re- arrangements that represent within-host micro-evolution during the 5-year niche adaptation in a human joint. As only two genome sequences are currently available for M. hominis, the comparative genomic analyses of these data to those generated for the arthritis strains and 12 additional clinical isolates will refine the core and pangenomes for the species, potentially revealing genes, gene islands or mobile genetic elements that are differentially associated with isolates from different disease pathologies. These data should also provide insight antigenic variation between different serotypes and the mechanism and acquisition of acquired tetracycline resistance. Ultimately, the data generated by completion of these Specific Aims should provide a platform from which strategies for improved immune or chemotherapeutic intervention can be devised and implemented.